Retinoblastoma, the most common intraocular tumor in children, is a prototype for studying the mechanism of tumor suppression. The identification of the RB gene and the substantiation of its role in cancer suppression marked a new era of cancer research. Based on these important discoveries, we propose three interrelated projects to further explore the biochemical and cellular functions of the Rb protein. The major aims are as follows: I) T o systematically analyze the structure/function relationship of Rb protein. Informative single point mutations in the C-terminal A and B domains of Rb will be identified by their ability to bind interacting proteins using reverse yeast two-hybrid method. Rb carrying these mutations will be characterized by assaying their ability to function in cell growth regulation and in mediating terminal differentiation. The biological function of the N-terminal domain of Rb will also be addressed using a transgenic approach to rescue the Rb-/- developmentally lethal phenotype, and to suppress pituitary tumor development. Key factors that interact with the N-terminal domain of Rb will also be identified. II) To determine if Rb plays a role in chromosome segregation during M phase progression by specifically marking the chromosomes and testing their behavior in embryonic fibroblasts with RB +/+, +/- or -/- genotype. Establishment of this novel function of Rb will be useful for understanding its role in cancer suppression. III) To elucidate the biological significance of three RB associated proteins, C5, C15, and C11. C5 is a co-activator of the thyroid hormone receptor, C15 is a novel leucine heptad repeats-rich protein with a critical role in M phase progression, and C11 is a novel LXCXE containing protein with transactivation activity. The biological consequence of Rb interacting with these proteins will be explored. Results of these studies will contribute significantly to a basic understanding of the genesis of retinoblastoma in particular and human oncogenesis in general.